CELLULAR AND MOLECULAR CHARACTERIZATION OF MESENCHYMAL STEM CELLS DERIVED FROM PLURIPOTENT STEM CELLS AND PROSPECTION OF APPLICATIONS IN REGENERATIVE MEDICINE

Abstract

<h3>Background</h3> Stem cells have been studied for their properties on tissue regeneration and functional recovery of injured organs. Mesenchymal stem cells (MSCs) are prominent in this context as they promote the increase of activity and viability of several cell types through distinct mechanisms. However, biological samples of MSCs bring great variability in behavior and viability while in culture (e.g., cell population heterogeneity, senescence). Therefore, it is necessary to establish a standardized protocol for the generation of high-quality stable cell cultures. In this sense, the use of pluripotent stem cells (PSCs) in derivation protocols for generation of MSC-like cells (PSC-MSCs) is an alternative, since PSCs maintain their characteristics consistently under suitable culture conditions. <h3>Methods</h3> Pluripotent stem cells (HES3) grown as cell clumps were cultured in ultralow attachment plates in aggregation medium for 24 h under hypoxia to form embryoid bodies (EBs). Mesodermal differentiation was induced in the EBs during 4 days when CD56 mesodermal marker was evaluated by FACS. Induced EBs were then plated into gelatin coatings and cultured in standard MSC medium containing FBS. After 4 passages the generated cells were evaluated for classical positive and negative MSC markers as defined by ISCT. <h3>Results</h3> Mesoderm induction was observed in induced EBs by CD56 expression (78%). During culture in FBS-containing medium, EBs attached and migrating cells presented fibroblast-like morphology. With progression of culture PSC-MSCs acquired epithelial-like morphology and slowly retrieved fibroblast-like morphology until passage 4 (P4). FACS analysis in P4 identified positive MSC markers CD90/CD140b in the majority of PSC-MSCs, and CD73/CD13 present in about one third of the cells. CD105 expression was not detected. Negative MSC markers CD34/CD11b/CD45/CD19/CD31/HLA-DR were not expressed by PSC-MSCs as well. About 26% of the cells expressed CD90 and CD73 simultaneously. Half of the analyzed cells still expressed CD56, indicating PSC-MSC may be less mature than adult MSCs. <h3>Conclusion</h3> PSC-MSC derivation protocol via mesodermal induction is feasible and the generated cells expressed positive MSC markers and lacked the negative ones. PSC-MSC were expanded <i>in vitro</i> for at least four passages. Further investigations will analyze other minimal criteria of MSC in the derived cells as trilineage differentiation potential, besides transcriptomic and karyotyping analysis.